PhosphoProteomics Service
Our PhosphoProteomics & PhosphoProtein Array platform offers high sensitivity detection and accurate quantitation of protein phosphorylation. Furthermore, customers can choose to target: 1) all 3 types of phosphorylation (pSer/pThr/pTyr) by 2D DIGE PhosphoProtein Array, 2) a specific type by 2D Phospho Western Blot. Phospho-protein enrichment is also offered as an option (but not required) in sample preparation. Here are some highlighted features of our PhosphoProteomics platform:
- Visualize phospho-protein: Phosphorylated proteins can be seen clearly with fluorescent phospho-tag
- Fast screening: Only phospho-proteins are tagged, allowing instant removal of un-phosphorylated proteins
- Low cost: Enrichment of phospho-protein is not required
- Quantify phosphorylation level: Accurate quantitation of phosphorylation level of each protein and phospho-ratio of the same protein between samples
- High sensitivity: Fluorescent phospho-tag offers sensitivity higher than mass spectrometry
- High resolution: Effective separation of phospho-proteins from their unmodified form
- Fast turnaround: Only takes 7-9 days.
Main applications:
- Kinase/Phosphatase substrate mapping
- Kinase/Phosphatase activator screening and identification (such as drug induced protein phosphorylation)
- Kinase/Phosphatase inhibitor screening and identification
- Signal transduction studies (regulation of protein functions by phosphorylation)
Project Designs based on your objectives
- A. Identify proteins differentially phosphorylated (all 3 types: Phospho-Ser, Thr & Tyr)
- B. Identify proteins differentially phosphorylated at Ser or Thr or Tyr by 2D Western Blot
- C. Identify proteins that are phosphorylated
A. Identify proteins differentially phosphorylated (all 3 types: Phospho-Ser, Thr & Tyr)
Study design for 2 samples (Control, Test)
- Gel-1: Control, Phospho-profiling
- Gel-2: Test, Phospho-profiling
- Gel-3: Control, Test
Image reports:
- Gel-1: Total Protein image, PhosphoProtein-image and overlay image of Total Protein/PhosphoProtein
- Gel-2: Total Protein image, PhosphoProtein-image and overlay image of Total Protein/PhosphoProtein
- Gel-3: Protein image of each sample and overlay of the 2 samples
Gel 1:
Control: Total Protein
Control: Phosphoprotein
Total Protein / Phosphoprotein
Gel 2:
Test: Total Protein
Test: Phosphoprotein
Total Protein / Phosphoprotein
Gel 3:
Control: Total Protein
Test: Total Protein
Control / Test
Quantitation report:
- Phospho-ratio: Gel-1 & 2 will provide phospho-signal of each phosphorylated spots and phospho-ratio between 2 samples
- Protein ratio: Gel-3 will provide the protein ratios
- Final Phospho-ratio: Calculated by adjusting Phospho-ratios with protein ratios
| Assigned ID | Phospho Ratio | Protein Ratio | Final Phospho Ratio |
|---|---|---|---|
| 1 | 3.11 | 0.92 | 3.39 |
| 2 | 4.23 | 0.94 | 4.48 |
| 3 | 4.37 | 0.85 | 5.11 |
| 4 | 6.16 | 0.92 | 6.71 |
| 5 | 4.55 | 1.06 | 4.29 |
B. Identify proteins differentially phosphorylated at Ser or Thr or Tyr by 2D Western Blot
Study design for 2 samples
- Gel-1: Control, Western Blot with anti-pSer or pThr or pTyr antibody
- Gel-2: Test, Western Blot with anti-pSer or pThr or pTyr antibody
- Gel-3: Control, Test
Image reports:
- Gel-1: Total Protein image, PhosphoProtein WB image and overlay image of Total Protein/PhosphoProtein
- Gel-2: Total Protein image, PhosphoProtein WB image and overlay image of Total Protein/PhosphoProtein
- Gel-3: Protein image of each sample and overlay of the 2 samples
Quantitation report:
- Phospho-ratio: Gel-1 & 2 will provide phospho-signal of phospho-spots and phospho-ratio between 2 samples
- Protein ratio: Gel-3 will provide the protein ratios
- Final Phospho-ratio: Calculated by adjusting Phospho-ratios with protein ratios
Examples of 2D Western Blot with Anti-pSer or Anti-Tyr
Example 1. Anti-Phospho-Tyrosine (pTyr) Western Blot
Jurkat cells were treated with serum starvation for 1 hour. Then serum was added to the medium and cultured for 4 hours. Proteins were extracted by 2D lysis buffer following Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (A). Second, proteins were transferred to the membrane and Western Blot was performed using mouse monoclonal anti-pTyr antibody (Clone PY20) and CF488-labeled goat anti-mouse Ig (Biotium, Hayward, California). The Western Blot image was scanned using Typhoon 9400 (B) .
A. Cell Lysate Total Protein
B: Anti-pTyr Western Blot
C. Total Protein / Anti-pTyr WB
Example 2. Anti-Phospho-Serine (pSer) Western Blot
Hela cells were treated with 1 mM sodium orthovanadate for 30 minutes. Proteins were extracted by 2D lysis buffer following Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (A). Western Blot was performed using mouse monoclonal anti-pSer antibody (Sigma) and CF488-labeled goat anti-mouse Ig (Biotium, Hayward, California). The Western Blot image was scanned using Typhoon 9400 (B).
A. Cell Lysate Total Protein
B: Anti-pSer Western Blot
C. Total Protein / Anti-pSer WB
Example 3. Anti-Phospho-Serine/Threonine/Tyrosine (pSer/pThr/pTyr) Western Blot
Hela cells were treated with 1 mM sodium orthovanadate for 30 minutes. Proteins were extracted by 2D lysis buffer following Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (A). Western Blot was performed using mouse monoclonal antibodies against pSer/pTyr/pThr (Sigma) and CF488-labeled goat anti-mouse Ig (Biotium, Hayward, California). The Western Blot image was scanned using Typhoon 9400 (B).
A. Cell Lysate Total Protein
B: Anti-pSer/pThr/pTyr WB
C. Total Protein / Anti-pSer/pThr/pTyr
C. Identify proteins that are phosphorylated
Study design for 1 sample
Gel-1: Cell lysate, Phospho-profiling or Phospho-WB
A. Cell Lysate Total Protein
B: PhosphoProtein
C. Total Protein / PhosphoProtein
Study design for 2 samples:
The phospho-signal is the combined effects of both samples. If you want to obtain phospho-signal for each sample, please use Study design A.
Gel-1: Normal, Drug treated, Phospho-profiling or Phospho-WB
Image report (for 2 samples):
Gel-1: Protein image of each sample and overlay of the 2 samples; Phospho-image of 2 samples and overlay image of Protein/Phospho
1. Normal
2. Drug Treated
3. PhosphoProtein Array
Normal / Drug Treated
Normal / PhosphoProtein Array
Normal / Treated / Phosphoprotein
Quantitation report:
- Protein ratios will be provided on differently expressed protein spots
- Phopsho-spots will be marked on the protein ratio table
PhosphoProteomics Applications
1. Drug induced phospho-protein change
Identify Phospho-protein markers due to drug treatment: 2D DIGE was performed on 2 samples (1. Control, 2. Drug treated), followed by phospho-profiling. Data showed that Phospho-protein level increased after drug treatment, while un-phospho protein levels remain similar between the 2 samples.
Control: Protein
Phosphoprotein
Protein / Phospho
Figure 1: Protein image, Phospho-image, and Protein/Phospho overlay image of Control sample.
Drug-treated: Protein
Phosphoprotein
Protein / Phospho
Figure 2: Protein image, Phospho-image, and Protein/Phospho overlay image of Drug-treated sample
Control + Drug-treated
Phosphoprotein
Control/Treated/Phospho
Figure 3: Protein image, Phospho-image, and Protein/Phospho overlay image of Control/Drug-treated. Spots circled in purple are phospho-spots up-regulated by drug treatment. Spots circled in white are Non-phospho-spots of the same protein which remain un-changed with drug treatment.
2. Phospho-protein time course
Identify phospho-protein changes in a time course: 2D DIGE and Phospho-profiling was performed on Embryonic stem (ES) cells differentiated at Days 0, 2, 4, 6, 8 and 10 (D0-D10). Data showed that Phospho-isoforms of Aconitate hydrase spots expressed at different levels during the time course.
Figure 1: Black / white image of Phosphorylated Aconitate hydrase (spot 1-3) and non-Phosphorylated Aconitate hydrase (spot 4) at each time point.
Figure 2: Color overlay image of each time point / D0.
Figure 3: Color overlay image of each time point / prior time point
Figures 1-3 indicate that Phosphorylated Aconitate hydrase (spot 1-3) changed over the time course, with the biggest change at Day 4 & 6. In contrast, non-Phosphorylated Aconitate hydrase (spot 4) remain at a similar level at different days. Please see Table 1 for protein ratios.
Table 1: Protein ratios of Aconitate hydrase (spot 1-4).
| Embryonic Stem Cell (ES) | D2 / D0 | D4 / D2 | D6 / D4 | D8 / D6 | D10 / D8 | |
|---|---|---|---|---|---|---|
| #1 | Phospho-Aconitate hydrase | -1.02 | -1.05 | +3.28 | -1.81 | -1.11 |
| #2 | Phospho-Aconitate hydrase | -1.41 | +1.41 | +3.54 | -1.96 | -1.06 |
| #3 | Phospho-Aconitate hydrase | -1.45 | +1.91 | +1.50 | -1.09 | -1.03 |
| #4 | Non-phospho-Aconitate hydrase | -1.45 | +1.91 | +1.28 | -1.07 | -1.03 |
Sample Info
Please follow these general principles regarding Biohazard material and buffer condition in getting your samples ready.
Protein amount: 500 -700 µg of total protein from each sample. If you order Phospho-Protein Enrichment (Item 450A and 450N), 5000 µg of total protein from each sample is needed. This will be sufficient to cover the entire procedure including Phospho-Protein Array, preparative gel, spot picking and protein ID.
Protein concentration: 5-20 mg/ml is preferred.
Buffer: Please feel free to submit samples in whichever buffer that you would normally use. The standard sample preparation is covered by the DIGE gel cost. Such samples should have protein concentration in the range of 5-20 mg/ml. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion.
Sample type: Please inform our scientists about your sample type. We will send you additional tips for each sample type.
Pricing
| Services Description | Academic/Governmental Labs | Industrial/Commercial Labs | ||
|---|---|---|---|---|
| Code | Price | Code | Price | |
| 2D Phospho-Protein Profiling 1 | 401A-PP1 | $1500 | 401N-PP1 | $1800 |
| 2D DIGE Phospho-Protein Profiling 1 | 401A-PP2 | $1900 | 401N-PP2 | $2280 |
| Phospho-Protein Enrichment (from 5mg) | 450A | $700 | 450N | $1000 |
1. Price covers:
- Experimental design
- Standard sample preparation and protein concentration determination
- Fluorescence dye labeling using CyDye
- IEF and SDS-PAGE
- Scanning of protein images using Typhoon scanner
- Phosphoprotein-staining of the same gel
- Scanning of protein/Phospho images using Typhoon scanner
- Two hour data analysis covering image and DeCyder analysis
- Data Report
Prices are discounted and only apply for customers doing follow up Protein ID service with Applied Biomics.
Price does NOT cover:
- Sample preparation that requires extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion
- Screen shots of 3D view of each spot
- Preparative gel, spot picking, protein ID: Please note that in most cases, Phospho-gels (similar to Analytical gels) do not contain sufficient amount of protein for protein identification. If you find some interesting spots you would like to move forward to perform Protein ID, we will need to run a Preparative gel. Please view here for differences between Analytical and Preparative gels. Please click here for the cost of Preparative gel, spot picking and protein ID
| Services Description | Academic/Governmental Labs | Industrial/Commercial Labs | ||
|---|---|---|---|---|
| Code | Price | Code | Price | |
| 2D DIGE Preparative Gel (1 CyDye labeling)* 1 | 401A | $839 | 401N | $999 |
| 2D DIGE Preparative Gel (2 CyDye labeling)* 1 | 102A2 | $1200 | 102N2 | $1500 |
| 2D DIGE Preparative Gel (3 CyDye labeling)* 1 | 102A3 | $1349 | 102N3 | $1649 |
| Spot Picking per Gel* 2 | 103 | $265 per 96 spots | 103 | $265 per 96 spots |
| Protein ID by Mass Spectrometry (MALDI-TOF/TOF) 3 | 104A | $139 per spot | 104N | $159 per spot |
| Pathways Analysis After Protein ID 4 | 203A | call for a quote | 203N | call for a quote |
*Price applies for customers who perform protein identification at Applied Biomics
1. Price covers:
- Experimental design
- Sample preparation and protein concentration determination
- Fluorescence dye labeling using CyDye (1 sample for 1 CyDye labeling; 2 samples for 2 CyDye labeling; 3 samples for 3 CyDye labeling)
- IEF and SDS-PAGE
- Gel image scan using Typhoon scanner
- Spot picking design
2. Price covers:
- Set up spot picking
- Pick spots
- Free storage at -80C for 6 months.
1-3. Prices are discounted and only apply for customers doing 2D DIGE & protein ID service with Applied Biomics. Turnaround time starting from Preparative gel to sending protein ID report is 7-10 business days. We offer high sensitivity protein identification from gel spots or bands using the latest technologies in mass spectrometry. In each run, four sensitivity standards in the amount of 1-10 femtomole are included. Our sensitivity is the range of 1-2 femtomole. Please view the protein ID procedure for detailed steps of this service. Price covers the following:
- Gel treatment
- In-gel trypsin digestion
- Peptide extraction
- Desalting
- Spotting
- MALDI-TOF
- MALDI-TOF/TOF
- Database search against NCBI database using MASCOT
- Data report (example below)
4. Pathway Analysis Service is provided only for customers doing the 2D DIGE and Protein ID services with Applied Biomics. The report will include the major pathway list with the following information:
- Functional groups
- Genes in each functional group
- Enrichment score
- Statistic p-value and FDR
